Project Summary Borrelia miyamotoi is a relapsing fever Borrelia group spirochete that is transmitted by the same hard-bodied (ixodid) ticks that transmit the agents of Lyme disease, anaplasmosis, and babesiosis. Human cases of B. miyamotoi infection were first reported in 2011 in Russia and subsequently in the United States, Europe, and Japan. These reports highlight the potential public health importance of human B. miyamotoi infection because it appears to be widespread and may cause severe disease, including meningoencephalitis. Clinical manifestations are non- specific so an etiologic diagnosis requires confirmation by blood smear examination, PCR, antibody assay, in vitro cultivation, and/or isolation by animal inoculation. Because the disease is not yet reportable, we have very little understanding of the prevalence of this infection, an important determinant of the health burden of disease. Population-based serosurveys have been used as a cost-effective and rapid method for initial determination of the prevalence of disease, however, the results of only two such B. miyamotoi studies have been published and these were focused on a limited area of southern New England. The antibody assay used in these serosurveys was a B. miyamotoi GlpQ antigen ELISA-Western blot assay that cannot distinguish between hard tick and soft tick relapsing fevers due to cross reactivity between relapsing fever Borrelia and is poorly suited for high-throughput evaluation of antibody status. Accordingly, we shall identify additional B. miyamotoi antigens using a proteomic approach to develop a new B. miyamotoi multiplex assay with improved sensitivity, specificity and automation compared with the current B. miyamotoi GlpQ assay. We shall use the B. miyamotoi multiplex assay and the standard Borrelia burgdorferi antibody assay in a population-based serosurvey method with a large sample size that is well suited for comparing the seroprevalence of B. miyamotoi infection and Lyme disease. Use of multiple study sites along East-West and North-South axes will allow us to determine the prevalence, geographic range, and emergence pattern of these two infections in the northeastern US. We also shall carry out case-finding to compare disease severity between B. miyamotoi infection and Lyme disease, as well as provide positive control sera for development of the B. miyamotoi multiplex assay. Such information is urgently needed because of the high level of human exposure to B. miyamotoi-infected ticks in some regions and the potential for this pathogen to cause severe illness with relapsing fever and central nervous system disease.